This research seeks to determine the role extracellular matrix (ECM) plays in the aging of skeletal muscle. The loss of skeletal muscle mass and strength that occurs during the aging process represents a major health issue facing a growing elderly population. Skeletal muscle homeostasis is the responsibility of the tissue's resident stem/progenitor cell known as the satellite cell or muscle progenitor cell (MPC). With age, the regenerative capacity of MAPCs is diminished. In determining how MPC regenerative capacity is lost, one must consider that both cell-intrinsic and cell-extrinsic changes can impact MPCs. Factors extrinsic to the cell include environmental components such as soluble factors, neighboring cells, and ECM, each of which can experience numerous changes with age. The research described here investigates the impact ECM age has on the ability of MPCs to carry out processes required for the regenerative maintenance of skeletal muscle tissue. We hypothesize that alteration of skeletal muscle ECM during the aging process results in misregulation of the regenerative mechanisms used by MPCs to maintain skeletal muscle homeostasis. This is among the first research to isolate the effects of ECM from cellular and soluble component effects on muscle regeneration as a function of age. In this proposed study, we will utilize ECM and MPCs from different ages of animals to study the interaction between them and the effect ECM has on progenitor cell function, as well as its impact on functional tissue formation. Rats have been chosen as the model organism to minimize genetic variability and gain precise control over the age of both the ECM and the MPCs. Using a method developed in our lab, skeletal muscle tissue from young adult (<12 months) and old rats (>24 months) will be decellularized, a process which removes cellular material while leaving ECM intact. The influence of ECM age on the growth behavior and regenerative capacity of young and old MPCs will be investigated using two in vitro models. In the first, ECM will be extracted from the decellularized tissue and used to coat tissue culture dishes onto which MPCs will be seeded. In the second, slices of decellularized tissue will be used as scaffolds onto which MPCs will be seeded and three-dimensional (3D) tissue will be grown under physiological loading conditions in a bioreactor system. In both culture systems, old or young rat MPCs will be seeded onto plates or scaffolds of old or young ECM in a two-by-two factorial experimental design. In both systems, the impact of ECM age on myogenesis, proliferation, and differentiation of the MPCs will be measured, including monitoring the expression of proteins known to be required for myogenic differentiation. Additionally, global gene expression will be assayed to determine how gene expression of MPCs is altered based on the age of the ECM component of the culture system. Finally, the function of MPC- scaffold constructs will be measured to determine how the formation of functional engineered tissue is affected by the age of the ECM component. When completed, this research will provide significant insight into the role ECM age plays in regulating the regenerative capacity of a stem/progenitor cell population vital to the health of elderly individuals. These data will provide the basis for future investigations of the compositional differences in the cell microenvironment that presents stimulatory and/or inhibitory cues to regenerative muscle cells. PUBLIC HEALTH RELEVANCE: The loss of skeletal muscle mass and strength characteristic of the aging process represents an important health problem facing a growing elderly population. A fundamental question that remains unanswered is the role that age-related changes to the physical microenvironment, or extracellular matrix, of muscle stem cells play in the aging of skeletal muscle. This research is designed to determine how an aging cellular microenvironment affects the regenerative capacity of skeletal muscle progenitor cells and to identify the genes and proteins whose expression and activity is adversely affected by age-related changes to the cell microenvironment.